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At least 19 recordsLinked to original sources
PubMed1998

Aphid species--vectors of plum pox virus.

Plum pox virus (PPV) is widely spread by natural vectors present in plum orchards. The efficiency of transmission is dependent on the frequency of the occurrence of vectors and the cultivar susceptibility to this pathogen. Having in view that PPV has a wide range of annual and multiannual host plants and vectors, there is great concern for obtaining PPV-resistant cultivars. This report deals with the following vectors: Hyalopterus pruni, Brachycaudus cardui, Brachycaudus helichrysi. Myzus persicae and Phorodon humuli aphids, and Aculus fockeni mite. Seven different cultivars of Prunus domestica were utilized. To assess the virus transmission rate, 50-100 individual vectors per tree were used. The treatment of the trees was performed every four weeks and then the disease symptoms were observed. PPV was transmitted by all vectors studied, the rate ranging in dependence on the susceptibility of cultivars used. Thus, in cvs. Centenar, Pescarus, d'Agen, Stanley and Tuleu Gras, the transmission rate ranged from 20% to 60%, while in susceptible cvs. Vanat romanesc and Vanat de Italia--from 40% to 80%.

Animals↗
PubMed1998

Production of a monoclonal antibody specific to the El Amar strain of plum pox virus.

Plum pox virus (PPV) isolates are grouped into three clusters differentiated by biological, serological, molecular and epidemiological characteristics: Marcus (M), Dideron (D) and Cherry (C). The El Amar (EA) isolate that does not fit any of the above groups is also known. Monoclonal antibodies (MAbs) that specifically recognize M, D, and C strains of PPV are already available. To complete the set of PPV strain-specific serological reagents, MAbs against the EA isolate were raised by immunizing BALB/c mice and fusing their spleen cells with NS0/1 myeloma cells. After a preliminary characterization by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), 1 of 13 selected MAbs proved to be EA strain-specific. This MAb (EA24) reacted equally well with a homologous antigen and several PPV isolates from Egyptian apricot trees, supporting the hypothesis of an additional specific PPV group. MAb EA24 did not react either with about a hundred PPV isolates belonging to the D and M groups or with PPV-SwC and PPV-SoC isolates belonging to the C group. The strain specificity of MAb EA24 was confirmed by Western blot analysis and immunoelectron microscopy. We conclude that there is now available a set of MAbs which are highly specific to the four currently known groups of PPV strains.

Animals↗
PubMed2000

Biotechnological aspects of plum pox virus.

Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.

Biotechnology↗
PubMed2004

Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions.

Plum pox virus (PPV) is one of the most devastating diseases of Prunus species. Since few sources of resistance to PPV have been identified, transgene-based resistance offers a complementary approach to developing PPV-resistant stone fruit cultivars. C5, a transgenic clone of Prunus domestica L., containing the PPV coat protein (CP) gene, has been described as highly resistant to PPV in greenhouse tests, displaying characteristics typical of post-transcriptional gene silencing (PTGS). We show in this report that C5 trees exposed to natural aphid vectors in the field remained uninfected after 4 years while susceptible transgenic and untransformed trees developed severe symptoms within the first year. C5 trees inoculated by chip budding showed only very mild symptoms and PPV could be detected in these trees by IC-RT-PCR. The PPV-CP transgene in C5 was specifically hyper-methylated with no detectable expression. These results indicate both stability and efficiency of PTGS-based PPV resistance in plum under field conditions.

Animals↗
PubMed2006

Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

Biomarkers↗
PubMed2001

Pathogenicity determinants in the complex virus population of a Plum pox virus isolate.

Several subisolates were separated from a single Plum pox virus (PPV) isolate, PPV-PS. In spite of an extremely high sequence conservation (more than 99.9% similarity), different subisolates differed largely in pathogenicity in herbaceous hosts and infectivity in woody plants. The severity of symptomatology did not seem to correlate with virus accumulation. Sequence analysis and site-directed mutagenesis demonstrated that single amino acid changes in the helper component (HC) protein caused a drastic effect on virus symptoms in herbaceous hosts and notably modified virus infectivity in peach seedlings. These results indicate that HC variation might play an important role in virulence evolution of natural plant virus infections. Moreover, the analysis of Potato virus X (PVX)-HC chimeras showed that the identified HC amino acid changes had parallel effects on the severity of symptoms caused by PPV and on HC-induced enhancement of PVX pathogenicity, indicating that HC functions in potyvirus symptomatology and in synergism with other viruses have overlapping determinants.

Amino Acid Substitution↗
PubMed2001

Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene.

Transgenic plums containing the plum pox potyvirus coat protein (PPV-CP) gene were inoculated with PPV. Infection was monitored by evaluating symptoms, ELISA, and IC-RT-PCR. Transgenic clone C5 was highly resistant to PPV during four years of testing and displayed characteristics typical of post-transcriptional gene silencing (PTGS), including a high level of transgene transcription in the nucleus, low levels of transgene mRNA in the cytoplasm, a complex multicopy transgene insertion with aberrant copies, and methylation of the silenced PPV-CP transgene. The PPV-CP transgene was also methylated in seedlings of C5 and these seedlings were resistant to PPV. Our results show, for the first time, that PTGS functions as a mechanism for virus resistance in a woody perennial species.

Blotting, Southern↗
PubMed1998

Characterization of plum pox virus isolates from Slovakia.

Plum pox virus (PPV) isolates from stone-fruit trees (plums, myrobalans, apricots and peaches) from orchards and gardens were characterized. To characterize their biological properties, several PPV isolates were transmitted by chip budding to GF 305 seedlings and mechanically to selected herbaceous test plants. The isolates differed in severity of infection, host range and symptomatology. A subgroup differentiation of 43 isolates from 22 localities of western and middle Slovakia was accomplished using reverse transcription-polymerase chain reaction (RT-PCR), immunocapture RT-PCR (IC-RT-PCR) and restriction analysis. These assays confirmed the presence of isolates belonging to PPV-M and PPV-D subgroups. PPV-M and PPV-D isolates were almost equally represented in tested samples. Tests of subgroup variability of PPV isolates from infected tolerant plum cultivars showed great predominance of PPV-M isolates.

DNA, Plant↗
PubMed1998

High resistance and control of biological risks in transgenic plants expressing modified plum pox virus coat protein.

Transgenic plums transformed with the plum pox virus coat protein (PPV CP) gene displayed a resistance to the sharka disease (Ravelonandro et al., 1997). However, the expression of PPV CP in transgenic plants may lead to complementation of deficient characteristic of an incoming potyvirus. Indeed, an aphid-intransmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) could be transmitted when encapsidated by the engineered PPV CP (Lecoq et al., 1993). To control such a risk, new PPV CP constructs were designed and introduced into Nicotiana benthamiana genome. In the first construct, the DAG amino acid triplet involved in the potyvirus aphid-transmission was deleted. The second construct encoded a truncated PPV CP lacking its first 140 amino acids. In the last construct, the nucleotides encoding the charged amino-acids R220, Q221 and D264 localized in the core of the PPV CP were removed. A bacterial expression system was developed to show that these deletions prevent the assembly of the PPV CP subunits. For each construct, several transgenic lines were produced and first challenged with several strains of PPV. Two phenotypes of resistance were observed: recovery and immunity. Their biochemical characterization showed that the resistance was RNA-mediated and therefore can be classified as homology-dependent (Jacquet et al., 1998a). Resistant lines producing high level of wild type or modified PPV CP were then inoculated with ZYMV-NAT to perform an aphid-transmission assay. Results of these experiments demonstrated that the use of modified forms of PPV CP genes in transgenic plants provide a good way to control the biological risks associated with heteroencapsidation (Jacquet et al., 1998b).

Animals↗
PubMed1998

New results concerning the plum pox virus epidemiology and resistance of plum cultivars, hybrids and rootstocks.

Our recent results on the plum pox virus (PPV) epidemiology show that PPV spreads very rapidly in plum tree plantations in the contaminated areas. A clearing of the PPV-infected trees reduces significantly the spread of the virus but does not eliminate the disease. Some plum tree cultivars, hybrids and rootstocks (Scoldus, Alina, Cristi, BN 1/8Fl, BN 2Gr. etc) showing field resistance could not be infected with PPV by natural way. However, they could be infected with PPV by artificial inoculation except for the plum tree cv. Local of Dragasani and the BN 4Kr myrobalan, which proved to be immune to PPV. PPV was not transmitted through seeds in plum tree and myrobalan in the nursery. The Hyalopterus pruni aphids were found PPV-positive by an enzyme-linked immunosorbent assay (ELISA).

Agriculture↗
PubMed1998

Characterization of phenotype resistance to plum pox of transgenic plums expressing plum pox virus capsid gene.

Resistance to plum pox virus (PPV) infection can be obtained in transgenic plants that express the virus capsid gene. An Agrobacterium-mediated transformation was used to introduce the PPV capsid gene into Prunus domestica plants. Over 11 regenerated plants (clones) were observed for the development of the disease symptoms and analysed for the presence of PPV by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) through 4 dormancy cycles. The level of protection against PPV was determined in the transformed plants, non-transformed plants, and a control transgenic plant "transformed" with the plasmid vector alone. One clone, C-5, appeared fully protected, while PT-6 and C-4 clones accumulated a low concentration of virus and the rest of the clones was entirely susceptible. Little is known about the mechanisms of resistance to virus infection in transgenic woody plants. To investigate this aspect, comparative studies based on the characteristics of resistant and susceptible clones have been started. A question, whether the phenotype resistance of clone C-5 is similar to that observed in transgenic herbaceous plants or not, has been addressed. Recent progress in this investigation is presented.

Capsid↗
PubMed2005

Aphid (Hemiptera: Aphididae) species composition and potential aphid vectors of plum pox virus in Pennsylvania peach orchards.

Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.

Animals↗
PubMed2004

A simplified RT-PCR-based detection of recombinant Plum pox virus isolates.

Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination between PPV-D and PPV-M have been recently identified and shown naturally spread in several European countries. As their serological properties were identical with those of conventional PPV-M isolates, they could be detected only by combined analysis of at least two different genome parts. To simplify the detection of such recombinants primers specific to PPV-M and PPV-D sequences with binding sites located on both sides of the recombination crossover situated in the C-terminal part of NIb were designed. They were used for direct differentiation of PPV-M, PPV-D and their recombinants by reverse transcription-polymerase chain reaction (RT-PCR). This method is convenient for identification of a recombinant PPV in single as well as mixed infection with PPV-M or PPV-D.

DNA Primers↗
PubMed1993

Comparative sequence analysis of four complete primary structures of plum pox virus strains.

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5' terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3' terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5' and 3' ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5' end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Molecular variability of Czech plum pox virus isolates.

Seventy-two Czech plum pox virus (PPV) isolates from different hosts were tested by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). In addition, the coat protein mobility and the RsaI restriction fragment length polymorphism (RFLP) pattern of reverse transcription-polymerase chain reaction-(RT-PCR) amplified coat protein (CP) gene fragment of the isolates were analysed. Both PPV-D and PPV-M serotypes were found in the Czech Republic. The results obtained by these different methods were in accord with exception of a few cases probably caused by mutation or recombination.

Antibodies, Monoclonal↗
PubMed2005

Accumulation of the long class of siRNA is associated with resistance to Plum pox virus in a transgenic woody perennial plum tree.

We investigated the hallmarks of posttranscription gene silencing (PTGS) in mature plants, embryos, and seedlings of the transgenic plum trees (Prunus sp.) that are resistant to Plum pox virus (PPV). We previously demonstrated that the transgene insert and resistance to PPV were mutually inherited in progeny of line C5. We show here that C5 constitutively produces a short (22 nt) and a long (25 to 26 nt) species of short interfering (si)RNA from embryo to mature plant in the absence of PPV inoculation. Unlike siRNA, methylation and transcription of the PPV-coat protein transgene were 're-set' following seed germination. Uninoculated transgenic susceptible clones did not display DNA methylation, nor did they produce detectable levels of siRNA. Upon infection, susceptible clones, transgenic or untransformed, did produce siRNA but only the short 22-nt species. These findings show that plum trees respond to virus infection by initiating PTGS-like mechanisms that involve the production of siRNA. We further suggest that high-level virus resistance in transgenic Prunus species requires the production of the long-size class of siRNA. The research adds new insights into PTGS silencing in woody perennial plant species.

Genes, Viral↗
PubMed1993

Monoclonal antibodies suitable for plum pox virus determination.

Monoclonal antibodies (MAbs) to plum pox virus (PPV) were obtained after immunization of BALB/c mice with purified PPV-W isolate. Spleen cells from a mouse showing a high serum titer were used for fusion with Sp2/0-Ag14 myeloma cells. Culture supernatants were screened for specific antibody production against PPV-W isolate using indirect ELISA. A total of six stable hybridoma lines producing MAbs of IgG class were obtained. All four PPV isolates tested (W, A, D and M) can be distinguished by these MAbs. Two highly efficient antibodies were chosen for practical purposes, and their applicability in PPV diagnostics has been studied since 1989 in parallel with polyclonal antibodies and commercial kits (Boehringer, Bioreba). The experiments have proven comparable sensitivity with a detection limit ranging between 10 and 50 ng of virus per ml sample. In routine diagnostics of plums, peaches and apricots our MAbs ranked at least as high as the commercial kits.

Animals↗